Detecting polynucleotide sequences

Abstract

A method of detecting a target polynucleotide sequence in a sample uses a labelled polynucleotide secondary probe, and a polynucleotide primary probe having sequences complementary to both the target and the secondary probe. The sample, immobilized or in solution is hybridized with the primary probe; and the labelled secondary probe is also hybridized with the primary probe. The method permits the production of a labelled secondary probe which can be used in conjuction with many different primary probes for different hybridization reactions.

I claim: 
     
       1. A method of detecting a specific target polynucleotide sequence in a sample, comprising the use of (a) a labelled polynucleotide secondary probe having a complex single-stranded polynucleotide sequence, and   (b) a polynucleotide primary probe having a single-stranded sequence complementary to the target sequence and a complex single-stranded sequence complementary to the complex sequence of the secondary probe,   which method comprises the steps of   (i) contacting the sample under hybridisation conditions with the primary probe,   (ii) before, during or after said contact hybridising the labelled secondary probe to the primary probe, and   (iii) observing the presence or absence of the label in association with the sample as indicating the presence or absence of the target sequence.   
     
     
       2. A method as claimed in claim 1, wherein the sample is immobilised on a solid support. 
     
     
       3. A method as claimed in claim 2, wherein the sample is contacted first with the primary probe and subsequently with the secondary probe. 
     
     
       4. A method as claimed in claim 2, wherein a solution of the secondary probe is first mixed under hybridising conditions with a solution of the primary probe and the immobilised sample is contacted with the resulting mixture. 
     
     
       5. A method as claimed in claim 3, wherein the primary probe is derived from a double-stranded DNA vector containing the single-stranded sequence complementary to the target which is denatured just prior to use. 
     
     
       6. A method as claimed in claim 4, wherein the primary probe is derived from a double-stranded DNA vector containing the single-stranded sequence complementary to the target which is denatured just prior to use. 
     
     
       7. A method as claimed in claim 2, wherein the primary probe is derived from a double-stranded DNA vector containing the single-stranded sequence complementary to the target, the secondary probe is derived from a corresponding double-stranded DNA vector which has been labelled, and a mixture of the two probes is first denatured and then added under hybridising conditions to the immobilised sample. 
     
     
       8. A method as claimed in claim 1, wherein a solution of the sample is contacted under hybridising conditions with a solution of a single-stranded primary probe, a solution containing partly double-stranded sequences but not single-stranded sequences is recovered and contacted under hybridising conditions with a solution of a labelled single-stranded secondary probe, remaining single-stranded sequences are removed, and the presence or absence of label in association with the sample is observed as indicating the presence or absence of the target sequence in the sample. 
     
     
       9. A method as claimed in claim 1, wherein the primary probe is based on a single-stranded plasmid or bacteriophage and the secondary probe is derived from a replicative double-stranded form of the bacteriophage. 
     
     
       10. A method as claimed in claim 1, wherein the primary probe is of DNA and the secondary probe is of RNA.